Problem with Acquity BEH Shield RP18

[Mattias]

I have developed a (beautiful) separation of a mixture of peptides on a Acquity BEH Shield RP18, but the separation does not work after approximately 20 injections of sample. The sample is just a mixture of nine pure peptides in water (MW about 1000), i.e. very clean solution.

Isocratic method, 20% acetonitrile in 10 mM unbuffered ammonium acetate.
Flow: 0.5 ml/min
Temp: 70C
Inj vol: 10 µl
Column: Acquity BEH Shield RP18, 1.7 µm, 2.1 * 100mm

After about 20 injections the peptides with acidic functions start to loose retention, messing up the separation. Normally you would think that the pH of the mobile phase has changed, but the separation is not improved by preparing new mobile phase. Changing to a new column is the only way to get the separation back again (new column + old mobile phase = OK)

Are my conditions to harsh for this type of column, or do you have any other ideas?

[Uwe Neue]

This version of the BEH particles is based on a monofunctional silane (the other ones are using trifunctional silanes). Thus its stability is lower, and I suspect that at 70 degrees and your pH you are stripping off the bonded phase.

Either go to the BEH C18, or try with this column an ammonium acetate buffer pH 4.5.

[Mattias]

You are probably right, but I am surprised that pH 6.8 and 70C is too harsh for a column that was designed to cope with "everything". The application notes says the working range for the Shield RP-18 column is pH 1-11, which is simply not true.

I will install a mobile phase heater before the column and reduce the temperature. Hopyfully a decrease in temperature from 70 to 60C can save the column.

Changing to another column means I need to start from scratch again, the selectivity of the BEH C18 is very different from the Shield RP-18.

[Mattias]

I decreased the temperature to 50C, and two peaks shifted elution order. The minimum resolution remained the same = OK.

I will let the system run over the weekend with the lower temperature (brand new column). If the separation is gone on Monday, I will be very disappointed in the BEH-technology. Even our old Lichrospher columns can handle these conditions.

[Mattias]

Well, now the system has been running for the whole weekend and the separation still looks OK on Monday morning. It is not 100% the same separation as a had Friday afternoon, but the differences are negligible (not affecting the minimum resolution).

Uwe told me that the RP-Shield columns are more sensitive to temperature than the other BEH-columns, which seems to be true. The shift from 70 to 50C made a huge difference in this case at least.

[Mattias]

I saw there was another post regarding drifting selectivity of the Acquity columns, so I thought I should wrap up the conclusions from my issue.

Waters confirmed that the Shield RP-18 contains some cation exchange groups from the production of the material. I could understand that these groups are not really wanted in the material, but more of a side-reaction. Apparantly all "Shield" columns contains these groups.

These groups are slowly released from the material during use, which changes the retention of acidic components in the sample. The effect is not huge, but clearly seen in my case (isocratic method, peptides with acidic side groups).

I can still run this separation, but I need to adjust the pH when the column is getting older (new column = pH 6.8, old column = pH 6.0).

[Mattias]

Sorry, it should read "anion-exchange groups"!